Peptide Inhibitors of Calcium Oxalate Monohydrate Crystallization and Uses Thereof

ABSTRACT

In an embodiment, the present disclosure pertains to a composition for inhibiting calcium oxalate monohydrate crystal growth comprising at least one isolated polypeptide comprising a plurality of amino acids that bind the surface of the calcium oxalate monohydrate crystal; and a plurality of amino acids spacers, wherein the amino acid spacers are arranged in varying sequences between the plurality of amino acids that bind the surface of the calcium oxalate monohydrate crystal. In some embodiments, the present disclosure related to a method of controlling calcium oxalate monohydrate crystal growth in a subject in need thereof comprising administering to the subject therapeutically effective amount of the calcium oxalate monohydrate inhibiting polypeptide. In some embodiments, the present disclosure relates to a method of identifying calcium oxalate monohydrate inhibiting peptides. Such a method may comprise designing a peptide library of potential calcium oxalate inhibiting peptides; screening the peptide library for high efficacy inhibitor peptides for inhibition of calcium oxalate monohydrate crystallization; and conducting molecular characterization of the high efficacy inhibitor to determine specificity.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/844,143, filed on Jul. 9, 2013, which is incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with U.S. government support under grant No.1207441 and No. 1207411 awarded by the National Science Foundation. TheU.S. government may have certain rights in this invention.

BACKGROUND

Calcium oxalate is the most common constituent of urinary calculi andrelatively large crystals of this salt are frequently found in freshlyvoided urine from patients with recurrent calcium-containing stones.Current treatments of Calcium Oxalate Monohydrate (COM) stone diseaseinclude water intake and diet supervision, which collectively reducecalcium oxalate supersaturation in urine. Hydrochlorothiazide, sodiumpotassium phosphate, and potassium citrate are drugs available for thetreatment of calcium oxalate stone disease and reported to reduce itsrecurrence. While these treatments can be effective, they do notsuppress stone incidence. Therefore, there is a need to develop moreeffective drugs for preventing calcium oxalate stone formation and todissolve kidney stones/fragments.

SUMMARY

In some embodiments the present disclosure provides a composition forinhibiting calcium oxalate monohydrate crystal growth comprising atleast one isolated polypeptide comprising a plurality of amino acidsthat bind the surface of the calcium oxalate monohydrate crystal; and aplurality of amino acids spacers that lack β-carbon side chains. In someembodiments the amino acid spacers are interspersed in varying sequencesbetween the plurality of amino acids that bind the surface of thecalcium oxalate monohydrate crystal.

In some embodiments, the present disclosure provides a method ofcontrolling calcium oxalate monohydrate crystal growth in a subject inneed thereof. In some embodiments, such a method comprises administeringto the subject a therapeutically effective amount of at least one of theaforementioned COM inhibitory polypeptides.

In another embodiment, the present disclosure pertains to a method ofidentifying calcium oxalate monohydrate inhibiting peptides. In someembodiments, such a method comprises the steps of designing a peptidelibrary of potential calcium oxalate inhibiting peptides. In a relatedembodiment, the method comprises screening the peptide library for highefficacy COM inhibitor peptides. In an embodiment, the method comprisesconducting molecular characterization of the high efficacy inhibitor.

In an embodiment, the present disclosure relates to a method ofinhibiting abnormal biomineralization in a subject in need thereof. Insome embodiments, such a method comprises administering to the subjecttherapeutic effective amounts of at least one of the aforementioned COMinhibitory polypeptides. In some embodiments, the abnormalbiomineralization causes disease selected from the group consisting ofrecurrent stone disease, primary hyperoxaluria, and systemic oxalosis.

The above objects and other objects, features, and advantages of thepresent invention are readily apparent from the following detaileddescription of the best mode for carrying out the invention when takenin conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

In order that the manner in which the above recited and other advantagesand objects of the invention are obtained, a more particular descriptionof the invention briefly described above will be rendered by referenceto specific embodiments thereof, which are illustrated in the appendedFigures. Understanding that these Figures depict only typicalembodiments of the invention and are therefore not to be consideredlimiting of its scope, the invention will be described with additionalspecificity and detail through the use of the accompanying Figures inwhich:

FIG. 1 shows a rational approach to design and screen peptides asinhibitors of COM crystallization using a high-throughput scheme tosynthesize and screen peptide libraries, characterize peptide inhibitorspecificity at the molecular level (e.g. using modeling, AFM, etc.) forthe most promising candidates, and apply information gained fromsystematic studies of peptide inhibition for designing new sets ofpeptide libraries;

FIGS. 2A-2B show Ca²⁺ ion-selective electrode (ISE) measurements of COMcrystallization with peptide D1 at different concentrations (FIG. 2A).The COM growth rate was measured as the slope (dCa/dt) of decreasingCa²⁺ concentration within the first 40 min of crystallization. FIG. 2Bshows a scatter plot of screened peptide inhibitors of COMcrystallization, categorized in regions of low (LI, <20%), moderate (MI,20 to 35%) and high (HI, >35%) inhibition for reducing the rate of COMcrystal growth (FIG. 2B). The percent reduction in COM growth rate wascalculated by comparing the ISE slopes of peptides and control using theformula, PercentReduction=[1−(dCa/dt)_(peptide)/(dCa/dt)_(control)]×100%. Data areaverages of 2 or 3 measurements (error bars equal two standarddeviations);

FIGS. 3A-3F show SEM images of COM crystals from bulk crystallization inthe presence of 20 μg/ml peptide inhibitors of the present disclosure.Control crystals (no peptide additive) exhibit an elongated hexagonalplatelet morphology with basal {100} surfaces bounded by {010} and {121}sides (FIG. 3A). COM crystals prepared with peptide D4 exhibit higherlength-to-width aspect ratio (FIG. 3B). Several peptides in the libraryalso induced rounding of the apical tip (arrow in B). Images takennormal to the {010} plane permit a comparison of COM {100} thicknessamong samples. It was observed that the control (FIG. 3C) hasapproximately twice the thickness as test peptide D7 (FIG. 3D). Peptidesthat bind to {121} surfaces produce diamond-shaped crystals with (FIG.3E) large {121} surface area and (FIG. 3F) small {121} surface area(shown for COM crystals prepared using peptides D11 and D1,respectively);

FIGS. 4A-4B show macroscopic characterization of COM crystals from bulkcrystallization in solutions of 0.5 mM CaC₂O₄ and 20 μg/ml peptide.Optical microscopy was used to measure: Percent distribution ofelongated hexagonal platelet (shaded bars) and diamond platelet (patternbars) crystal morphologies (FIG. 4A); and length of the COM (100) basalsurface measured along the [001] direction (i.e. apical tip-to-tipdistance) for hexagonal and diamond morphologies (FIG. 4B). Data in(FIG. 4A) and (FIG. 4B) are the average of three separatecrystallization experiments performed at 60° C. for 3 days (error barsequal one standard deviation). Data for peptide D4 (diamond habit) wasomitted based on too few crystals for statistical analysis;

FIG. 5 shows inhibition of COM crystal growth as evaluated by kineticstudies of COM growth at 60° C. using peptides E10 and E11 (20 μg/mL);

FIGS. 6A-6B show bulk crystallization studies of COM in presence ofselect peptides. Peptides E6-E10 produced COM crystals with longerdimensions as compared to control (FIG. 6A). An increased aspect ratiorelative to the control was also observed with various peptides (FIG.6B);

FIG. 7 shows a polydisperse distribution of crystals produced peptidesE6 and E7. Many of the crystals possessed the elongated hexagonal shapethat is characteristic of control crystals, while others had adiamond-shape that is consistent with peptide binding to surfaces.Peptide E6 was less effective than E7, as suggested by the moderateeffects on crystal morphology observed in scanning electron micrographs(e.g. a rounding of the apical tips was noticed in some cases, FIG. 7);and

FIG. 8 shows a comparison of the effects of Asp-Ala peptides and Glu-Alapeptides on COM crystal aspect ratio. The aspect ratio (c/b) comparesthe length-to-width ratio of COM platelets along the [001] and [010]directions, respectively.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are not restrictive of the invention, as claimed. In thisapplication, the use of the singular includes the plural, the word “a”or “an” means “at least one”, and the use of “or” means “and/or”, unlessspecifically stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements or components comprising one unit and elements orcomponents that comprise more than one unit unless specifically statedotherwise. Parameters disclosed herein (e.g., temperature, time,concentration, etc.) may be approximate.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated herein byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials defines a termin a manner that contradicts the definition of that term in thisapplication, this application controls.

Crystallization is ubiquitous in biological systems where interactionsbetween inorganic (salt, ions, etc.) and organic components (proteins,lipids, etc.) often mediate physiological processes in the human body,such as bone and teeth formation [1, 2]. Under abnormal physiologicalconditions, mineralization can lead to such pathologies asatherosclerotic plaques or vascular calcifications, kidney orgallstones, gout, and osteoarthritis. Small molecules that inhibitabnormal biomineralization are potentially effective therapies againstsuch conditions.

Kidney stone disease is a common pathological disorder that affects morethan 10-15% of the U.S. population with incidence rates that are on therise [3, 4]. Kidney stone pathogenesis is a complex process thatinvolves a series of steps operating either singularly orsynergistically to produce polycrystalline aggregates in the kidney [5].Calcium oxalate monohydrate (COM) is the most common component of humankidney stones. Supersaturated calcium oxalate in urine facilitates COMcrystal nucleation and growth. The aggregation of COM crystals and thecumulative retention of crystals and/or aggregates in the kidney haveadverse effects once stones reach an appreciable size and becomedislodged from epithelial membrane. Inhibiting one or more of thecritical pathways of COM stone pathogenesis nucleation, growth,aggregation, and retention via the addition of external agents canpotentially serve as an effective therapy for this disease.

The rate of crystal growth and aggregation of calcium oxalate willdetermine whether or not a particle large enough to be trapped at somenarrow point in the urinary tract can be formed within the transit timeof urine through the urinary system. The chance of a particle beingtrapped depends partly on the size of the particle. The rate of crystalgrowth may also determine to a large extent the subsequent rate ofgrowth into a stone. It has been proposed that crystal growth inhibitorspossess two types of moieties, a binder that strongly interacts withcrystal surface sites, and a perturber that sterically hinders theattachment of solute to crystal surfaces [6]. A common binder group ofCOM crystal inhibitors (i.e. urinary proteins and their syntheticanalogues) is carboxylic acid, which binds to oxalate vacancies on COMcrystal surfaces via calcium bridges, _((COM))COO⁻ . . . Ca²⁺ . . .⁻OOC_((inhibitor)). Lahav, Leiserowitz, and coworkers [7] have proposedmechanisms of crystal growth inhibition that occur through theadsorption of small molecules to surfaces of crystals growing byclassical nucleation and spreading of layers (so called layer-by-layergrowth). Inhibitors that bind to different sites on a crystal surface(i.e. steps, ledges, and terraces) reduce step advancement normal tothat surface. Inhibitors can therefore serve to retard crystal growth,with implications in therapies for biomineralization-based diseases, oralter growth rates of specific faces, with implications in crystal shapeengineering for design of advanced materials.

There are several native proteins in urine that are putative inhibitorsof COM crystal growth and/or aggregation [8, 9]. A common trait of theseinhibitors is an appreciable quantity of negatively-charged amino acids,L-aspartic acid (Asp, D) and L-glutamic acid (Glu, E), andphosphorylated or glycosylated modified groups in their structure.Polymeric macromolecules have been shown to be significantly more potentinhibitors of COM crystallization than their corresponding monomers[10], which can be attributed to proximal sites on the polymer chain(e.g. carboxylic acids) that cooperatively bind to COM crystal surfaces.

Past studies have investigated COM crystallization in the presence ofsynthetic molecules that mimic the functional moieties of proteininhibitors found in vivo, including small organic molecules, such ascitrate [11], and macromolecules, such as polyamino acids (poly-L-Aspand poly-L-Glu) and poly(acrylic) acid [12]. It has been shown thatthese peptides are effective in altering in vitro biomineralization[13]. For example, previous studies of COM crystallization have examinedthe effect of peptide mimics of urinary protein segments, notablyosteopontin (OPN), and showed that the amino acid sequence plays animportant role in determining peptide binding affinity to COM crystalsurfaces [14]. It has been suggested that OPN-derived peptides inhibitCOM growth by forming clusters or continuous films on COM crystalsurfaces [15], which can promote the formation of less stable hydrates(e.g. calcium oxalate dihydrate) [16]. Additional factors affecting theinhibition of COM crystallization include peptide phosphorylation [17,18], and the concentrations of peptide [18] and solute [19]. Similarobservations have been reported for other biominerals, such as calciumcarbonate [20] and calcium phosphate [21]. Studies using calcium-bindingpeptides rich in acidic amino acids observed that a number of propertiesof peptides influence crystallization, among which include peptidesubdomains [22], amino acid sequence [23] and length [24], and motifs(i.e. repeating Asp and Glu patterns) [25].

Peptides are an attractive template for designing tailored crystalgrowth inhibitors. The modular synthesis of peptides is amenable forhigh-throughput analyses, and permits modifiers to be constructed withcontrolled size, programmable sequences, secondary structure, andstereochemical modularity whereby residues can be easily substituted tosystematically alter chemical functionality and spatial proximity ofrecognition sites. They also allow for the rational design ofphysico-chemical properties, such as high solubility, highbioavailability, and low or no toxicity. Designing de novo sequencesfrom first principles is challenging due to the vast diversity in thechemical space and number of possible sequences of peptides.High-throughput methods that allow rapid synthesis and screening ofpeptide libraries can significantly enhance peptide design based onrational principles.

A challenge in the rational design of inhibitors is tailoring themolecular recognition between inhibitor and crystal through thejudicious selection of inhibitors with appropriate functionality, size,and structure. The present disclosure relates to the influence ofchemical functionality in small peptides derived from varying sequencesof Aspartic acid (Asp) and Alanine (Ala) amino acids. The rationale,philosophy and approaches of the present disclosure are translatable todesign of peptide modifiers (i.e. inhibitors and promoters) of crystalgrowth and properties, such as crystal habit and size.

Peptides are also an attractive template for engineering chemical andstructural motifs that provide effective crystal growth control. Thepresent disclosure pertains to the efficacy of short peptide sequences(˜18 amino acids in length) as effective COM crystallization inhibitors.Furthermore, the present disclosure relates to subtle changes to COMpeptide inhibitor sequence that can translate into profound changes ininhibitory potential of the peptides. Additionally, the presentdisclosure pertains to the use of ISE-based high-throughput screeningand identification of potent peptides, and their validation as effectiveinhibitors in bulk crystallization studies.

It is reasonable to expect that the application of the design algorithm,disclosed herein, using biomimetic peptide sequences derived fromtargeted segments of known calcium-binding proteins has the potential tofurther improve peptide performance as potent inhibitors of COM crystalgrowth. Furthermore, molecular-level analysis addressing fundamentalaspects of peptide-crystal interactions may establish an improvedunderstanding of structure-function properties for input into the designalgorithm for refinement and testing.

The versatile approach presented here has broader applicability for avariety of inorganic and organic materials. Notably, the resultsdisclosed herein are promising in their direct impact on kidney stonetherapies. In an exemplary embodiment, the COM inhibitors disclosedherein may be used to treat the great majority of patients sufferingfrom recurrent stone disease, since over 75% of all renal stones containcalcium oxalate. For example, the COM inhibitors may be useful toprevent formation of stones by preventing or inhibiting nucleation,growth and/or aggregation of crystals. The COM inhibitors disclosedherein may also be used to treat these patients following extracorporealshockwave lithotripsy to help ensure passage in the urine of shatteredstone particles and renal crystal deposits. The COM inhibitors of thepresent disclosure may also be effective in treating patients withprimary hyperoxaluria (a genetic disease resulting in massiveover-production of oxalic acid) many of whom suffer total loss of renalfunction in the early years of life. In additional embodiments, the COMinhibitors disclosed herein may have a potential use in dissolvingrecurrent renal stones in patients with a history of calcium oxalatestone disease. This could result in less frequent use of thelithotripter and other techniques now used to remove kidney stones. TheCOM inhibitors of the present disclosure may also have use in treatingpatients post renal transplantation in order to prevent calcium oxalatedeposition in the renal graft since many of these patients havesubstantial body stores of calcium oxalate following long-term dialysis.Finally, it is also envisioned that the COM inhibitors may be used totreat patients suffering from systemic oxalosis, i.e., deposition ofcalcium oxalate crystals in many tissues of the body. The stones beingtreated (or the formation of which is to be prevented) may be present inthe kidney, bladder and/or urinary tract. In a further embodiment, it isalso possible that other forms of treatment can be used in conjunctionwith administration of the compound of the present invention, i.e.,ultrasound treatment to break up the stones can be utilized on a patientwho has been treated with the COM inhibitors disclosed herein. In afurther embodiment, COM inhibitors of the present disclosure may beutilized in preventing calcification on implantable devices,prosthetics, or the like.

The COM inhibitors disclosed herein may be used for the treatment and/orprevention of calcium oxalate stone disease by administeringtherapeutically effective amounts of the inhibitor to a subject in needthereof. As used herein, the term “Subject” includes animals and humansrequiring intervention or manipulation due to a disease state, treatmentregimen or experimental design. For laboratory experiments, laboratorymammals such as rats, mice, monkeys, as well as other mammals can beused. However, the ultimately desired use is to treat human patients whosuffer from calcium oxalate stone disease.

It is contemplated that the COM inhibitor peptides will be formulatedinto a pharmaceutical composition comprising a therapeutically effectiveamount of the peptides with or without a pharmaceutically acceptablecarrier. The term “therapeutically effective” refers to reduction inseverity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of damage. Thepharmaceutically acceptable carrier may be any carrier which isnon-toxic, i.e., safe for human intake and which is compatible with theCOM inhibitor peptides and the desired route of administration. In aparticular embodiment of the disclosure, the therapeutic orpharmaceutical composition comprises the COM inhibitor in an effectiveunit dosage form. As used herein, the term “effective unit dosage” or“effective unit dose” means a predetermined amount of the COM inhibitorsufficient to be effective for dissolution of calcium oxalate kidneystones in vivo or effective to prevent or reduce the degree of formationof kidney stones in vivo.

The pharmaceutical compositions may be administered orally orparenterally, including by injection, subcutaneously or used as asuppository or pessary. The only limitation on the route ofadministration is that the COM inhibitor should reach the kidneys in anamount effective to treat kidney stone disease. It is also contemplatedthat it may be desirable for the COM inhibitor to reach the urinarytract or bladder of the patient being treated.

In some embodiments the present disclosure pertains to a composition forinhibiting calcium oxalate monohydrate crystal growth. Such acomposition comprises at least one isolated polypeptide comprising aplurality of amino acids that bind the surface of the calcium oxalatemonohydrate crystal and a plurality of amino acids spacers. In someembodiments of the present disclosure the amino acid spacers arearranged in varying sequences between the plurality of amino acids thatbind the surface of the calcium oxalate monohydrate crystal. In someembodiments the isolated polypeptide comprises at least 18 amino acids.

In some embodiments of the present disclosure the plurality of aminoacids that bind to the surface of the calcium oxalate crystals compriseL-Aspargine (D) and the plurality of amino acids spacers comprise aminoacids lacking the β-carbon side chains. In some embodiments, theplurality of amino acids spacers comprise L-Alanine (A). In someembodiments, the isolated polypeptide is selected from the groupconsisting of the amino acid sequences DDDAAAAADDDAAAAADD (SEQ ID NO.1), AADAAAAADDAAAADAAA (SEQ ID NO. 2), ADAAADAADAADDAADAA SEQ ID NO. 3,ADAADAADAADAADAADA (SEQ ID NO. 4), ADAADDAADAADDAAAAA (SEQ ID NO. 5),ADAAADDDAAADAAADDD (SEQ ID NO. 6), ADAAADDAAAAAAAADAA (SEQ ID NO. 7),ADAAADDAAADAAAADAA (SEQ ID NO. 8), ADAAADDAAADAAADDAA (SEQ ID NO. 9),ADAADAAADAADDAADAA (SEQ ID NO. 10), ADAADDAAAAAADAADAA (SEQ ID NO. 11),ADDAADAADAADDAADDA (SEQ ID NO. 12), and ADADADADADADADADAD (SEQ ID NO.13).

In some embodiments the plurality of amino acids that bind to thesurface of the calcium oxalate crystals comprise Glutamic acid (E) andthe plurality of amino acids spacers comprise Alanine (A) amino acids.In some embodiments, the isolated polypeptide is selected from the groupconsisting of the amino acid sequences AAEAAAAAEEAAAAEAAA (SEQ ID No.14), AEAAAEAAEAAEEAAEAA (SEQ ID NO. 15), AEAAEAAEAAEAAEAAEA (SEQ ID No.16), AEAAAEEAAAEAAAAEAA (SEQ ID No. 17), AEAAAEEAAAEAAAEEAA (SEQ ID No.18), AEAAEAAAEAAEEAAEAA (SEQ ID No. 19), AEAAEEAAAAAAEAAEAA (SEQ ID No.20), AEEAAEAAEAAEEAAEEA (SEQ ID No. 21), AEEAEEAEEAEEAEEAEE (SEQ ID No.22), AEAEAEAEAEAEAEAEAE (SEQ ID No. 23), EEEEEEEEEEEEEEEEEE (SEQ ID NO.24).

Any amino acid in the above sequences may be replaced by an isomer oranalog of a conventional amino acid (e.g., a D-amino acid), non-proteinamino acids, post-translationally modified amino acids, enzymaticallymodified amino acid, a construct or structure designed to mimic an aminoacid. In an embodiment of the present disclosure a kinase can be used tophosphorylate the COM inhibitory peptide at biologically relevant sites.In a further embodiment golcosylation may be utilized. As a non-limitingexample, modified terminal groups of sugar residues that are believed tobe important in stone prevention, such as carboxylic acid moieties ofsialic acid groups, may be utilized.

In some embodiments, the COM inhibitory peptides of the presentdisclosure may be glycosylated. In some embodiments, the COM inhibitorypeptides of the present disclosure may be glycosylated with one or moreglycans. In some embodiments, the glycans may include, withoutlimitation, N-linked glycans, O-linked glycans, glycosaminoglycans, andcombinations thereof. In some embodiments, the glycans may includecarboxylic acid moieties. In some embodiments, the carboxylic acidmoieties may be utilized in stone prevention.

In more specific embodiments, the COM inhibitory peptides of the presentdisclosure may be glycosylated with a glycan that includes a sialicacid. In some embodiments, the carboxylic acid moieties on the sialicacid may be utilized in stone prevention.

In some embodiments the COM inhibitory polypeptides further comprise atleast one pharmaceutical carrier. A pharmaceutically acceptable carriercan additionally contain physiologically acceptable compounds that act,for example, to stabilize or increase the absorption of the COMinhibitory polypeptide to be administered. Such physiologicallyacceptable compounds include but are not limited to, for example,carbohydrates such as glucose, sucrose or dextrans; antioxidants such asascorbic acid or glutathione; chelating agents' such as EDTA, whichdisrupts microbial membranes; divalent metal ions such as calcium ormagnesium; low molecular weight proteins; lipids or liposomes; or otherstabilizers or excipients. COM inhibitory polypeptides can also beformulated with a material such as a biodegradable polymer or amicropump that provides for controlled slow release of the molecule.

In some embodiments the present disclosure relates to a method ofcontrolling calcium oxalate monohydrate crystal growth in a subject inneed thereof. Such a method comprises administering to the subject atherapeutically effective amount of at least one of the aforementionedCOM inhibitory polypeptides. A therapeutically effective amount of a COMinhibitory polypeptide to be administered is any amount deemed nontoxicbut sufficient to inhibit calcium oxalate crystal growth in the subjectin need thereof.

In some embodiments the at least one COM inhibitory polypeptide isselected from the group consisting of the amino acid sequencesDDDAAAAADDDAAAAADD (SEQ ID NO. 1), AADAAAAADDAAAADAAA (SEQ ID NO. 2),ADAAADAADAADDAADAA SEQ ID NO. 3, ADAADAADAADAADAADA (SEQ ID NO. 4),ADAADDAADAADDAAAAA (SEQ ID NO. 5), ADAAADDDAAADAAADDD (SEQ ID NO. 6),ADAAADDAAAAAAAADAA (SEQ ID NO. 7), ADAAADDAAADAAAADAA (SEQ ID NO. 8),ADAAADDAAADAAADDAA (SEQ ID NO. 9), ADAADAAADAADDAADAA (SEQ ID NO. 10),ADAADDAAAAAADAADAA (SEQ ID NO. 11), ADDAADAADAADDAADDA (SEQ ID NO. 12),and ADADADADADADADADAD (SEQ ID NO. 13).

In some embodiments the isolated COM inhibitory polypeptide is selectedfrom the group consisting of the amino acid sequences AAEAAAAAEEAAAAEAAA(SEQ ID No. 14), AEAAAEAAEAAEEAAEAA (SEQ ID NO. 15), AEAAEAAEAAEAAEAAEA(SEQ ID No. 16), AEAAAEEAAAEAAAAEAA (SEQ ID No. 17), AEAAAEEAAAEAAAEEAA(SEQ ID No. 18), AEAAEAAAEAAEEAAEAA (SEQ ID No. 19), AEAAEEAAAAAAEAAEAA(SEQ ID No. 20), AEEAAEAAEAAEEAAEEA (SEQ ID No. 21), AEEAEEAEEAEEAEEAEE(SEQ ID No. 22), AEAEAEAEAEAEAEAEAE (SEQ ID No. 23), EEEEEEEEEEEEEEEEEE(SEQ ID NO. 24).

In some embodiments, the subject has kidney stone disorder. In someembodiments, the subject has renal calcification disorder. In someembodiments, the subject has biomineralization induced disease. In someembodiments, the method further comprises using ultrasound therapy.

In some embodiments, the present disclosure relates to a method ofidentifying calcium oxalate monohydrate inhibiting peptides. In someembodiments such a method comprises the steps of designing a peptidelibrary of potential calcium oxalate inhibiting peptides. In someembodiments, the method further comprises screening the peptide libraryfor high efficacy inhibitor peptides for inhibition of calcium oxalatemonohydrate crystallization. In some embodiments the method comprisesconducting molecular characterization of the high efficacy inhibitor todetermine specificity.

In some embodiments of the present disclosure, the step of designing apeptide library comprises selecting an amino acid that acts as a binderto bind to the surface of the COM crystal. In some embodiments, themethod further comprises selecting an amino acid that acts as a spacerto minimize the steric hindrance of the amino acid binder to COM crystalsurface and synthesizing peptides by varying the number of spacer aminoacids between the binder amino acids.

A COM inhibitory polypeptide of the present disclosure may be preparedor obtained by methods known in the art including, for example,purification from an appropriate biological source or by chemicalsynthesis. In addition to synthesis, inhibitory polypeptides may beproduced, for example, by enzymatic or chemical cleavage of largersequences. Methods for enzymatic and chemical cleavage and forpurification of the resultant protein fragments are well known in theart (see, for example, Deutscher, Methods in Enzymology, Vol. 182,“Guide to Protein Purification,” San Diego: Academic Press, Inc. (1990),which is incorporated herein by reference).

Following synthesis and purification, the COM inhibitory polypeptidescan be modified in a physiologically relevant manner by, for example,further phosphorylation, acylation or glycosylation, using enzymaticmethods known in the art.

The COM inhibitory peptide of the present disclosure can also berecombinantly expressed by appropriate host cells including, forexample, bacterial, yeast, amphibian, avian and mammalian cells, usingmethods known in the art. Methods for recombinant expression andpurification of peptides in various host organisms are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York (1992) and in Ansubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md. (1989), both of which are incorporated herein by reference. In someembodiments, the peptides are synthesized using solid-phase peptidesynthesis.

In some embodiments, the step of screening the peptide library for highefficacy inhibitor peptides comprises mixing the test peptide with asupersaturated solution of calcium oxalate and measuring inhibition ofgrowth of the COM crystal using in situ calcium ion-selective electrodemeasurement.

In some embodiments, the molecular level characterization of theidentified peptide is by Atomic Force Microscopy, Scanning ElectronMicroscopy, and Optical Microscopy.

In some embodiments, the present disclosure relates to a method ofinhibiting abnormal biomineralization in a subject in need thereofcomprising administering to the subject therapeutic effective amounts ofthe aforementioned COM inhibitory polypeptides.

In some embodiments the at least one COM inhibitory polypeptide isselected from the group consisting of the amino acid sequencesDDDAAAAADDDAAAAADD (SEQ ID NO. 1), AADAAAAADDAAAADAAA (SEQ ID NO. 2),ADAAADAADAADDAADAA SEQ ID NO. 3, ADAADAADAADAADAADA (SEQ ID NO. 4),ADAADDAADAADDAAAAA (SEQ ID NO. 5), ADAAADDDAAADAAADDD (SEQ ID NO. 6),ADAAADDAAAAAAAADAA (SEQ ID NO. 7), ADAAADDAAADAAAADAA (SEQ ID NO. 8),ADAAADDAAADAAADDAA (SEQ ID NO. 9), ADAADAAADAADDAADAA (SEQ ID NO. 10),ADAADDAAAAAADAADAA (SEQ ID NO. 11), ADDAADAADAADDAADDA (SEQ ID NO. 12),and ADADADADADADADADAD (SEQ ID NO. 13).

In some embodiments the isolated COM inhibitory polypeptide is selectedfrom the group consisting of the amino acid sequences AAEAAAAAEEAAAAEAAA(SEQ ID No. 14), AEAAAEAAEAAEEAAEAA (SEQ ID NO. 15), AEAAEAAEAAEAAEAAEA(SEQ ID No. 16), AEAAAEEAAAEAAAAEAA (SEQ ID No. 17), AEAAAEEAAAEAAAEEAA(SEQ ID No. 18), AEAAEAAAEAAEEAAEAA (SEQ ID No. 19), AEAAEEAAAAAAEAAEAA(SEQ ID No. 20), AEEAAEAAEAAEEAAEEA (SEQ ID No. 21), AEEAEEAEEAEEAEEAEE(SEQ ID No. 22), AEAEAEAEAEAEAEAEAE (SEQ ID No. 23), EEEEEEEEEEEEEEEEEE(SEQ ID NO. 24).

In some embodiments, the abnormal biomineralization causes diseaseselected from the group consisting of recurrent stone disease, primaryhyperoxaluria, and systemic oxalosis.

Additional Embodiments

Reference will now be made to various embodiments of the presentdisclosure and experimental results that provide support for suchembodiments. Applicants note that the disclosure herein is forillustrative purposes only and is not intended to limit the scope of theclaimed subject matter in any way.

Example 1

COM crystals for bulk crystallization studies were synthesized using areported procedure that produces micron-sized crystals with large basal(100) surfaces. COM crystals prepared with molar composition 0.7 mMCaCl₂: 0.7 mM Na₂C₂O₄: 150 mM NaCl are referred to as the control.Growth solutions prepared with peptides used 20 μg/ml of the additive(unless otherwise stated). The crystals were characterized by opticalmicroscopy using a Leica DM2500-M microscope, scanning electronmicroscope (SEM) using a FEI 235 Dual-Beam Focused Ion-beam instrument,atomic force microscopy (AFM) using an Asylum MFP-3D-SA instrument(Santa Barbara, Calif.), and a calcium ion-selective electrode (ISE,ThermoScientific).

Peptides were synthesized on an automated peptide synthesizer (MultipepRS, Intavis Inc., Germany). Using solid-phase peptide synthesis (SPSS)chemistry, peptides were synthesized from their C-termini to N-terminion tentagel amide resin (Intavis Inc.). Post synthesis, the peptideswere cleaved from the resin. Post-cleavage, the peptides werelyophilized and stored as dry and lyophilized powders for subsequentuse.

Example 2 Designing a Peptide Library for Screening COM GrowthInhibitors

Peptides provide a unique template for designing COM growth inhibitorsdue to an unparalleled ability to synthesize sequences with controlledsize, chemical functionality, spatial patterning, and secondarystructure. De novo peptides can be designed and tested, although theinfinite number of combinations calls for a more rational approach toselect lead candidates. To this end, Applicants searched for inspirationamong proteins that mediate biomineralization of calcium crystals(oxalates, carbonates, and phosphates). Calcium-binding proteins tend tobe rich in L-aspartic acid (Asp, D) and L-glutamic acid (Glu, E), butexhibit a wide variety of primary amino acid sequences with differentperiodicity (e.g. XDX, XDDX, XDDDX, etc.) that cannot be uniquelyidentified a priori as being the most effective for COM crystalinhibition. As such, Applicants used a simple design for creating apeptide library employing one binder, L-Asp, and one spacer (perturber),L-Ala. The selection of the binder was based on a general observationthat protein inhibitors of COM are rich in Asp and Glu. Applicants choseAsp for these studies as a representative example. Ala was chosen as aspacer (perturber) based on the lack of β-carbons (i.e. small sidegroup, R═CH₃) to minimize steric hindrance of L-Asp binding to COMsurfaces. Moreover, the hydrophobic residue of Ala may promotepeptide-COM binding via entropic effects wherein oriented watermolecules surrounding the methyl group are released when Ala orients ona COM crystal surface. This effect has been proposed for antifreezeprotein (AFP) inhibition of ice crystallization in cold-weather species(e.g. fish, plants, and insects) [27]. In brief, AFPs contain many Thrgroups with H-binding residues that promote AFP-ice adhesion. It hasbeen suggested that Ala groups (located in close proximity to Thr) are asecondary binder that promote AFP adsorption via an entropic effectattributed to the release of unfavorable hydration layers whenhydrophobic CH₃ groups bury into vacancies on ice surfaces [28].

Table 1 lists the peptide sequences selected for these studies (named D1to D13), which represent combinations of randomly selected Ala-Aspsequences. Some of these peptides possess similar XDX, XDDX, and XDDDX,patterns with subtle changes, such as the removal or addition of asingle binder or spacer group. This library was used as an initial testfor the design approach to synthesize and screen effective inhibitors ofCOM crystallization. Details of the design platform and experimentalscreening of this peptide library are presented in the followingsections.

TABLE 1 Peptide library synthesized for high-throughput studies of COMgrowth inhibition. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 D1 D D DA A A A A D D D A A A A A D D D2 A A D A A A A A D D A A A A D A A A D3A D A A A D A A D A A D D A A D A A D4 A D A A D A A D A A D A A D A A DA D5 A D A A D D A A D A A D D A A A A A D6 A D A A A D D D A A A D A AA D D D D7 A D A A A D D A A A A A A A A D A A D8 A D A A A D D A A A DA A A A D A A D9 A D A A A D D A A A D A A A D D A A D10 A D A A D A A AD A A D D A A D A A D11 A D A A D D A A A A A A D A A D A A D12 A D D AA D A A D A A D D A A D D A D13 A D A D A D A D A D A D A D A D A D

Example 2 High-Throughput Platform for Screening Peptide Inhibitors

The schematic in FIG. 1 outlines the high-throughput platform proposedhere for the design, testing, and modeling of peptide inhibitors of COMcrystallization. This sequential approach employs facile analyticalmethods to rapidly quantify macroscopic changes in COM crystal growthrate and bulk crystal habit. Techniques used in this study monitor thetemporal evolution of Ca²⁺ supersaturation during COM growth and thefinal bulk crystal morphology, which permits fast and reproducibleassessment of peptide specificity and efficacy. The overarching goal ofthe design platform in FIG. 1 is to refine large libraries of peptidesto a list of the most effective inhibitors for molecular-level studiesof peptide-crystal interactions that are typically time and effortintensive. These include, for instance, scanning force microscopy [29]and molecular modeling [30] that have proven effective for investigatingadsorbate interactions at solid-liquid interfaces. Applicants anticipatethat the application of this high-throughput platform in future COMstudies will provide valuable information of peptide-crystal molecularrecognition as an input for the design of new libraries. This process ofpeptide synthesis, screening, and systematic studies constitutes aniterative optimization loop wherein molecular-level investigation ofselect inhibitors can be used to develop heuristic guidelines forfurther design and refinement of peptide inhibitors.

Example 3 Screening the Peptide Library for High Efficacy Inhibitors ofCOM

A quantitative comparison of peptide efficacy for inhibiting COMcrystallization was performed using in situ calcium ion-selectiveelectrode (ISE) measurements. Supersaturated solutions of calciumoxalate (S=5.4) with peptide (20 μg/ml) were stirred to minimize theinduction period of crystal nucleation (observed times ranged from 0 to20 min with stirring). The temporal depletion of Ca²⁺ (ppm) during COMgrowth is approximately linear within the first hour of measurement, asshown in FIG. 2A for ISE potency measurements of COM crystallization atvarying concentrations of test peptide D1. This study revealed that 20μg/ml peptide in COM growth solutions was sufficient to observe COMgrowth inhibition with statistical certainty. As such, measurementsreported herein were conducted at a single peptide concentration (20μg/ml) to assess COM growth inhibition. To facilitate comparison ofpeptides in Table 1, Applicants calculated the percent reduction in COMgrowth rate using the relative difference in ISE slopes (units of ppmCa²⁺ per time) for peptide and control samples during the first 40 minof crystallization. A scatter plot of ISE results in FIG. 2B reveals alarge distribution of peptide efficacy spanning 0.6 to 58% reduction inCOM growth rate.

Some of the most potent inhibitors of COM reported by other groups aremacromolecules, such as OPN or poly(aspartic) acid, which reduce COMgrowth by >90% [12]. Small molecules generally tend to be less effectiveinhibitors of COM crystallization. A notable exception is citrate, whichis a small organic molecule with three carboxylic acid groups. Citrateis a moderately effective inhibitor used as an oral therapy for humanstone disease [11]. Here Applicants analyzed citrate as a benchmark fordetermining the relative effectiveness of peptides as COM growthinhibitors. ISE measurements of COM crystallization with 20 μg/mlcitrate yielded a 28±6% reduction in COM growth rate, which iscomparable to the average percent inhibition observed for all peptidesin Table 1. Interestingly, more than 30% of the peptides performedbetter than citrate in this small library based on crude designprinciples. Using the results of citrate as a reference point,Applicants subdivided data in FIG. 2B into three regions of low (LI),moderate (MI), and high (HI) inhibition, where the threshold for aneffective inhibitor was defined as any peptide exhibiting >35% reductionin COM growth rate (i.e. statistically higher than citrate), and thosewith low efficacy exhibiting <20% reduction.

Inhibitor performance from ISE screening should be evaluated within thecontext of COM growth conditions—notably temperature, calcium oxalatesupersaturation, inhibitor concentration, and ionic strength. It isimportant to mention that ISE, while shown here to be an effectivemethod for quickly and reproducibly screening large peptide libraries,does have its limitations for assessing COM crystallization for detailedkinetic studies near equilibrium. The sensitivity of ISE electrodesnecessitates the use of high (non-physiological) calcium oxalateconcentration. Many groups have focused on COM growth (with and withoutinhibitors) using CaC₂O₄ concentrations near equilibrium. For instance,Wang et al. reported 48% reduction in COM growth rate using a similarcitrate concentration as our study, but much lower supersaturation(S=1.3) [11]. It is reasonable to expect that high efficacy peptidesidentified in ISE analyses may exhibit more pronounced inhibition of COMgrowth rates at lower supersaturation (i.e. conditions that mimic COMcrystallization in vivo).

The slope of ISE curves (FIG. 2A) is the temporal change in free Ca²⁺concentration due to COM nucleation (primary and secondary) and crystalgrowth. Deconvoluting these two processes (nucleation and growth) fromISE data is nontrivial, particularly if one wishes to extract detailedkinetic information of COM growth rates. To this end, Applicants suggestusing an alternative approach, such as the constant composition (CC)method pioneered by Nancollas and coworkers [18]. In the current study,the distinct advantage of ISE compared to more traditional approaches isits ease of use and the rapid time for data acquisition that enableshigh-throughput analysis of large peptide libraries.

Example 4 Influence of Peptide Sequence on COM Growth Inhibition

Close inspection of trends in FIG. 2B reveals a nontrivial relationshipbetween peptide efficacy and its sequence (Table 1). Elucidating themechanism of peptide-COM recognition would require the use of moresophisticated techniques (e.g. molecular simulations, scanning probemicroscopy, etc.) to probe molecular-level details of peptideinteractions with COM crystal surfaces. The goal of this study is tovalidate a platform capable of screening a large number of potentialgrowth inhibitors in a reasonably short period of time to identify“hits” for more systematic, fundamental studies. Without a mechanisticunderstanding of peptide-crystal molecular recognition, it is difficultto draw definitive conclusions here. Nevertheless, there are severalinteresting observations in FIG. 2B that emphasize how subtle changes inpeptide sequence influence its efficacy as a COM growth inhibitor. Incomparing peptides D4, D5, and D9, which contain identical numbers ofL-Asp groups but different sequences, Applicants observe that thearrangement of L-Asp groups has a pronounced effect on the percentreduction in COM growth rate. The ADA sequence of peptide D4 yielded a20% reduction in COM crystal growth. The rearrangement of binders togenerate a mixture of D and DD sequences (peptide D5) increased thepercent reduction to 30%; and further alteration of this sequence byinserting one L-Ala spacer between each D or DD group (peptide D9)further enhanced peptide efficacy to achieve a 40% reduction in COMgrowth. If Applicants also compare the results of peptides D7, D8, andD9, it was observed that the sequential replacement of one L-Ala spacerwith one L-Asp binder increased peptide efficacy by a factor of ˜70.Among the library of peptides in Table 1, peptide D7 was the leasteffective inhibitor of COM crystallization (<1% reduction in COMgrowth). Substitution of an L-Ala with L-Asp at the 11^(th) amino acidposition yields peptide D8, which exhibited an order of magnitudeincrease in efficacy (˜6% reduction). A second substitution at the15^(th) amino acid position yielded peptide D9, which exhibited an evenfurther increase in efficacy (˜40% reduction).

Designing peptide inhibitors from de novo principles is challenging. Forinstance, an extensive study of an 18-mer peptide using all naturalamino acids would be virtually impossible. An exhaustive study of allunique 18-mer sequences derived from a library of only L-Asp and L-Alaamino acids would require screening a smaller, but still substantiallylarge, library of more than 10⁵ peptides. A more rational approach wouldbe the use of sequences from known protein inhibitors of COMcrystallization as a starting point for peptide design. Indeed, paststudies by DeYoreo [31] and Hunter [14] identified potent inhibitors ofCOM growth using peptide mimics of OPN segments. DeYoreo and coworkersexamined COM growth in the presence of 27-mer peptides with repeatingDDDX sequences (where X=Ser or Gly space groups). They reported a 90%reduction in COM growth using peptide concentrations of 0.02 μg/ml(X=Gly) and 2.2 μg/ml (X=Ser), where the 30-fold increase in peptidepotency was achieved by simply switching the spacer from glycine (Rgroup=H) to serine (R group=CH₂OH). DeYoreo and Hunter also testedsmaller 14-mer [18] and 16-mer [32] segments of OPN, respectively, andshowed that phosphorylation of primary amino acid sequences can achievemore than 60% reduction in COM growth.

Example 5 Bulk Crystallization Studies to Assess Peptide-COM Specificity

ISE measurements can be used to screen peptide efficacy, but providelittle information regarding the specificity of peptide binding todifferent surfaces of COM crystals. To this end, Applicants used asecond analysis step to identify the effect of peptides on COM crystalsize and habit. The influence of the peptide library on macroscopicproperties of COM crystals was assessed using bulk crystallizationstudies and optical and scanning electron microscopy to characterize COMcrystals grown in the presence of peptides. In vitro, COMcrystallization yields elongated hexagonal platelets with {100} basalsurfaces bounded by {010} and crystallographically equivalent {121}apical surfaces (FIG. 3A). Crystals prepared in the absence of peptide(control, FIG. 3A) exhibit basal surfaces with a ˜35 μm length along theaxis, ˜13 μm width along the [010] axis, and a thickness of ˜9 μm alongthe [100] axis (see FIG. 3C). Applicants observed that multiple peptidesin the library reduced growth along the [121] directions, whichdecreased the crystal aspect ratio (length-to-width) and shifted themorphology from hexagonal to diamond platelets. This change in crystalhabit can be attributed to the preferential binding of peptides to COM{121} surfaces, which slows growth along the naturally fast growthdirections within the (100) plane. This result is qualitativelyconsistent with the reduced growth rates reported in Section 3.3.

Two distinct crystal habits of diamond shape were observed in bulkcrystallization studies. The most commonly observed shape had large{121} surface area (FIG. 3D), while the least common shape had smaller{121} surface area (FIG. 3E) that closely matched the control.Interestingly, peptide-COM interactions did not significantly roughenbasal (100) surfaces, as might be expected from past studies ofinhibitor-COM interactions [33]. To more systematically assess thesurface topography of COM crystals, Applicants used atomic forcemicroscopy (AFM) to probe the effect of peptides on COM (100) surfaceroughness. Crystals extracted from control and peptide D5 batches wereprepared on AFM sample disks and imaged in air. The RMS roughness for(100) surfaces of hexagonal-shaped crystals from the control and testpeptide D5 were approximately 1.0 and 4.3 nm, respectively. The RMSroughness of diamond-shaped crystals from the peptide study was 3.2 nm,which is comparable to the hexagonal crystal for peptide D5, but largerthan the control. Despite the increase in RMS roughness, AFM images (notshown) revealed little difference in surface topography of crystalsprepared with and without peptide.

Electron micrographs revealed that select peptides from the library inTable 1 reduced the [100] thickness of COM crystals, which suggests apreferential interaction of these peptides with the COM {100} surface.Notably, peptide D7 decreased the [100] thickness of hexagonal plateletsby nearly a factor of two (FIG. 3D), while most other peptides had onlymarginal effect on platelet thickness. Another interesting observationwas that a majority of the peptides induced rounding of the apical tipformed by the intersection of {121} planes (arrow in FIG. 3B) onhexagonal platelets. The SEM image in FIG. 3B is a representativeexample of tip rounding, although in select cases Applicants did observemore pronounced rounding than shown here.

COM crystals prepared from peptide solutions were divided into twopopulations based on their habit—diamond and hexagonal platelets—wherethe relative percentage of diamond-shaped crystals is related to peptideefficacy and specificity for binding to COM {121} surfaces. FIG. 4Acompares the percentage of diamond and hexagonal COM crystals for thepeptide library (data for each peptide is an average of three separatebulk crystallization experiments). All peptides produced diamond-shapedcrystals, but the majority exhibited less than 20% diamonds in theirtotal crystal population. Peptides D5 and D8 exhibited the largestpopulations of diamond-shaped crystals, which suggests these peptidesbind more effectively to COM {121} surfaces and inhibit growth along the[121] directions. Applicants tested the effect of doubling theconcentration of peptide D8 (i.e. increasing the concentration from 20to 40 μg/ml), but Applicants did not observe an appreciable increase inthe population of diamond crystals.

Crystal batches prepared in bulk studies were analyzed using opticalmicroscopy to quantify changes in COM crystal size and aspect ratio.Diamond-shaped COM crystals exhibited an average [001] length of ˜20 μmand a length-to-width aspect ratio of 1.63±0.05, which is smaller thanthe aspect ratio of control crystals (2.67±0.05). Although there wereclear differences in the percent population of diamond-shaped crystalsamong peptides tested in this study, Applicants observed that the sizeof diamond crystals were generally the same, irrespective of peptidesequence (FIG. 4B). The majority of COM hexagonal platelets in peptidesamples had similar size and aspect ratio as the control (hereApplicants only report the length of COM crystals in FIG. 4B). A notableexception, however, was peptide D4, which produced a 22% increase in thelength-to-width aspect ratio of COM hexagonal crystals, and an increasein the [001] length from 35 μm (control) to 75 μm (peptide D4). Thissuggests that peptide D4 exhibits a preferential interaction with COM(010) surfaces. To a lesser extent, peptides D6 and D1 also increasedthe [001] length of hexagonal COM crystals by factors of 1.5 and 2.0,respectively, relative to the control.

The embodiments described herein are to be construed as illustrative andnot as constraining the remainder of the disclosure in any way. Whilethe embodiments have been shown and described, many variations andmodifications thereof can be made by one skilled in the art withoutdeparting from the spirit and teachings of the invention. Accordingly,the scope of protection is not limited by the description set out above,but is only limited by the claims, including all equivalents of thesubject matter of the claims. The disclosures of all patents, patentapplications and publications cited herein are hereby incorporatedherein by reference, to the extent that they provide procedural or otherdetails consistent with and supplementary to those set forth herein.

Example 6 Design of Peptides with Glutamic Acid Binding Moieties

A second library of peptides containing glutamic acid (E) and alanine(A) amino acids was synthesized. The sequences of E-peptides shown inTable 2 were used in bulk crystallization studies and kinetic studies toassess their role as growth inhibitors or promoters of COMcrystallization. Glutamic acid possesses a carboxylic acid group thatcan form a calcium bridge with surface oxalate groups of COM crystals.Calcium ISE measurements of COM growth at room temperature in thepresence of peptides E3, E5, and E6 revealed a percent inhibition in therange 30 to 50% (see Table 2). Kinetic studies of COM growth at 60° C.using peptides E10 and E11 (20 μg/mL) also revealed inhibition of COMcrystal growth (FIG. 5). These analyses were performed using the calciumassay to track the kinetics of crystallization over a 24-hour period.The calcium ion concentration in the supernatant solution was assessedat periodic times. Peptide E10 was a more effective inhibitor than E11.

Bulk crystallization studies of COM in the presence of select peptidesrevealed that many E-peptides preferentially bind to the {121} surfacesof COM crystals. This results in an increase in the [001]/[010] aspectratio relative to the control (FIG. 6). Peptides E6 to E10 produced COMcrystals with longer [001] dimensions than control crystals. Moreover,the peptides produced a polydisperse distribution of crystals, as shownin FIG. 7 for peptides E6 and E7. Many of the crystals possessed theelongated hexagonal shape that is characteristic of control crystals,while others had a diamond-shape that is consistent with peptide bindingto {121} surfaces. Peptide E6 was less effective than E7, as suggestedby the moderate effects on crystal morphology observed in scanningelectron micrographs (e.g. a rounding of the apical tips was noticed insome cases, FIG. 7).

TABLE 2 Peptide library synthesized withglutamic acid (E) and alanine (A) amino acids. % Inhi- Peptide Sequencebition E1 A-A-E-A-A-A-A-A-E-E-A-A-A-A-E-A-A-A E2A-E-A-A-A-E-A-A-E-A-A-E-E-A-A-E-A-A E3A-E-A-A-E-A-A-E-A-A-E-A-A-E-A-A-E-A 40 ± 17 E4A-E-A-A-A-E-E-A-A-A-E-A-A-A-A-E-A-A E5A-E-A-A-A-E-E-A-A-A-E-A-A-A-E-E-A-A 36 ± 21 E6A-E-A-A-E-A-A-A-E-A-A-E-E-A-A-E-A-A 52 ± 16 E7A-E-A-A-E-E-A-A-A-A-A-A-E-A-A-E-A-A E8A-E-E-A-A-E-A-A-E-A-A-E-E-A-A-E-E-A E9A-E-E-A-E-E-A-E-E-A-E-E-A-E-E-A-E-E E10A-E-A-E-A-E-A-E-A-E-A-E-A-E-A-E-A-E E11E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E Control — 0.00

Example 7 Colorimetric Calcium Depletion Assay

Free calcium concentration in the crystallization solution was measuredby a colorimetric assay using Quantichrome DICA 500 Calcium assay kit(BioAssay Systems, Hayward, Calif.). Standard solutions (0, 0.1, 0.2,0.3, 0.4, 0.5, and 1 mM) were prepared using calcium solutions providedwith the kit. Detection reagent was prepared by mixing equal volumes ofreagent A and reagent B according to manufacturer's instructions. A 10μL sample was withdrawn from the top of each crystallization wellwithout disturbing the bottom and dispensed into a 96-well microplate.100 μL of detection reagent was added to each well and incubated for 3minutes at room temperature. The absorbance was measured at 612 nm usingBiotek Synergy H4 microplate reader (BioTek, Winooski, Vt.). Calciumconcentration was determined using a calibration curve prepared fromstandard calcium solutions.

Example 8 Evaluation of Crystal Growth Modifying Potential

Time course of depletion of free calcium in the crystallizationsolution, normalized to the initial concentration, was fitted to anonlinear logistic function using OriginPro Software (Origin Lab,Northhampton, Mass.). The logistic function relating calciumconcentration to time is given by

$\begin{matrix}{\lbrack{Ca}\rbrack_{t} = {A_{2} + \frac{A_{1} - A_{2}}{1 + \left( \frac{t}{t_{1/2}} \right)^{p}}}} & (1)\end{matrix}$

where [Ca]_(t) is the normalized calcium concentration at time t, A₁ isthe normalized initial supersaturation concentration (i.e at t=0), A₂ isthe normalized equilibrium concentration (i.e. at t=∞), p is the hillcoefficient which represents slope of the curve at midpoint, and t_(1/2)is the crystallization half-time (CHT), defined as the time at whichcrystallization is 50% complete (i.e. [Ca]_(t) _(1/2) =(A₁−A₂)/2). A₁was fixed at 1, and A₂, p and t_(1/2) were obtained from fitting thedata to equation 1. CHT was used as a measure of inhibition potentialsof the peptides. A one-way ANOVA in combination with Tukey post hoc testat p<0.05 level was used to probe statistical significance. Applicants'studied the effects of experimental parameters including volume, shakingand temperature on crystallization in designing a high-throughputscreening assay. Applicants have shown the logistic model effectivelyrepresents the three phases: induction, crystal growth and equilibriumobserved in our setup, and CHT (t_(1/2)) can be used as a measure ofefficacy of potential crystal growth modifiers. The peptide libraryscreened comprised of Asp and Ala residues (peptides D1-D13) atoptimized assay conditions of 37° C., crystallization volume of 600 μLin a shaking environment. The lead candidates D1, D9 and D13 identifiedby the high-throughput assay in this study agree with the potentinhibitors from ion selective electrode (ISE) studies. A single timepoint measurement at 6 hour was shown to be of effective use to evaluatethe inhibition potential of modifiers, and thus increase the throughputof the assay. The high throughput of the assay provides the opportunityto assess the growth modulation potential of crystal growth modifiers atvarying modifier concentrations, supersaturations and ionic strengthswith relative ease. The lead candidates identified from the screeningcan be characterized in depth for studying the effect of modifiers oncrystal morphology using microscopy techniques and using molecularsimulation studies to understand the modifier—crystal interactions.Finally, the assay can be translated to other crystallization systems ina relatively straightforward fashion for rational design and discoveryof growth modifiers for specific applications.

TABLE 1 Crystallization half-time (CHT), standard error (SE), and residual fit of the modelin equation 1 to the results of the high-throughput colorimetric calcium depletion assay for peptides D1-D13. CHTSE in Peptide Sequence (hr) CHT R³ D1 DDDAAAAADDDAAAAADD 6.482 0.8680.924 D2 AADAAAAADDAAAADAAA 3.533 2.005 0.91 D3 ADAAADAADAADDAADAA 4.1420.468 0.953 D4 ADAADAADAADAADAADA 4.432 0.749 0.903 D5ADAADDAADAADDAAAAA 4.641 0.361 0.972 D6 ADAAADDDAAADAAADDD 5.041 0.5680.947 D7 ADAAADDAAAAAAAADAA 5.771 0.474 0.971 D8 ADAAADDAAADAAAADAA4.792 1.29 0.791 D9 ADAAADDAAADAAADDAA 7.277 0.365 0.983 D10ADAADAAADAADDAADAA 6.054 0.651 0.951 D11 ADAADDAAAAAADAADAA 5.767 1.2310.859 D12 ADDAADAADAADDAADDA 7.135 0.321 0.989 D13 ADADADADADADADADAD8.114 0.659 0.964 Control 4.18 0.783 0.926

Example 9 Comparison of Glu and Asp Peptide Libraries on COMCrystallization

A comparison between five Asp-Ala peptides and five Glu-Ala peptideswith comparable sequences on COM crystal morphology is shown in FIG. 1.Preliminary results indicate that the Glu-Ala peptides and Asp-Alapeptides have nearly identical effects on COM crystal habit.

A quantitative comparison of peptide efficacy for inhibiting COMcrystallization was performed using in situ ISE measurements. Thepercent inhibition in COM growth rate was calculated using the relativedifference in ISE slopes in the presence of 20 μg/mL peptide. The ISEresults reveal that there is a nontrivial relationship between peptideefficacy and its sequence. The effect of Asp with Glu in peptidesequences was measured on COM growth inhibition (Table 2). Results fromthese studies show that peptides with identical sequences (but differentacidic amino acid groups) have different effects on COM growth. Forinstance, there is a notable difference between the effect of Asp-Alapeptides and Glu-Ala peptides on COM growth inhibition. Among the threepeptide sequences compared in Table 2, the glutamic acid substation(X=E) is more effective than the aspartic acid groups (X=D).

TABLE 2 Comparison of Glu-Ala and Asp-Ala peptides efficacy % inhibitionSequence X = D X = E AXAAXAAXAAXAAXAAXA  21 ± 10 40 ± 18AXAAAXXAAAXAAAXXAA 36 ± 8 36 ± 21 AXAAXAAAXAAXXAAXAA 36 ± 5 52 ± 16

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What is claimed is:
 1. A composition for inhibiting calcium oxalatemonohydrate crystal growth comprising: at least one isolated polypeptidecomprising: a plurality of amino acids that bind the surface of thecalcium oxalate monohydrate crystal; and a plurality of amino acidsspacers, wherein the amino acid spacers are arranged in varyingsequences between the plurality of amino acids that bind the surface ofthe calcium oxalate monohydrate crystal.
 2. The composition of claim 1further comprising at least one pharmaceutical carrier.
 3. Thecomposition of claim 1, wherein the isolated polypeptide comprises about18 amino acids.
 4. The composition of claim 1, wherein the plurality ofamino acids that bind to the surface of the calcium oxalate crystalscomprise L-Aspargine (D) and the plurality of amino acids spacerscomprise amino acids lacking the β-carbon side chains.
 5. Thecomposition of claim 5, wherein the plurality of amino acids spacerscomprise L-Alanine (A).
 6. The composition of claim 4, wherein theisolated polypeptide is selected from the group consisting of the aminoacid sequences DDDAAAAADDDAAAAADD (SEQ ID NO. 1), AADAAAAADDAAAADAAA(SEQ ID NO. 2), ADAAADAADAADDAADAA SEQ ID NO. 3, ADAADAADAADAADAADA (SEQID NO. 4), ADAADDAADAADDAAAAA (SEQ ID NO. 5), ADAAADDDAAADAAADDD (SEQ IDNO. 6), ADAAADDAAAAAAAADAA (SEQ ID NO. 7), ADAAADDAAADAAAADAA (SEQ IDNO. 8), ADAAADDAAADAAADDAA (SEQ ID NO. 9), ADAADAAADAADDAADAA (SEQ IDNO. 10), ADAADDAAAAAADAADAA (SEQ ID NO. 11, ADDAADAADAADDAADDA (SEQ IDNO. 12), and ADADADADADADADADAD (SEQ ID NO. 13).
 7. The composition ofclaim 1, wherein the plurality of amino acids that bind to the surfaceof the calcium oxalate crystals comprise Glutamic acid (E) and theplurality of amino acids spacers comprise Alanine (A) amino acids. 8.The composition of claim 7, wherein the isolated polypeptide is selectedfrom the group consisting of the amino acid sequences AAEAAAAAEEAAAAEAAA(SEQ ID No. 14), AEAAAEAAEAAEEAAEAA (SEQ ID NO. 15), AEAAEAAEAAEAAEAAEA(SEQ ID No. 16), AEAAAEEAAAEAAAAEAA (SEQ ID No. 17), AEAAAEEAAAEAAAEEAA(SEQ ID No. 18), AEAAEAAAEAAEEAAEAA (SEQ ID No. 19), AEAAEEAAAAAAEAAEAA(SEQ ID No. 20), AEEAAEAAEAAEEAAEEA (SEQ ID No. 21), AEEAEEAEEAEEAEEAEE(SEQ ID No. 22), AEAEAEAEAEAEAEAEAE (SEQ ID No. 23), EEEEEEEEEEEEEEEEEE(SEQ ID NO. 24).
 9. A method of controlling calcium oxalate monohydratecrystal growth in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of the composition ofclaim
 1. 10. The method of claim 9, wherein the subject has kidney stonedisorder.
 11. The method of claim 9, wherein the subject has renalcalcification disorder.
 12. The method of claim 9, wherein the subjecthas biomineralization induced disease.
 13. The method of claim 9,further comprising using ultrasound therapy.
 14. A method of identifyingcalcium oxalate monohydrate inhibiting peptides comprising the steps of:designing a peptide library of potential calcium oxalate inhibitingpeptides; screening the peptide library for high efficacy inhibitorpeptides for inhibition of calcium oxalate monohydrate crystallization;and conducting molecular characterization of the high efficacy inhibitorto determine specificity.
 15. The method of claim 14, wherein the stepof designing a peptide library comprises: selecting an amino acid thatacts as a binder to bind to the surface of the COM crystal; selecting anamino acid that acts as a spacer to minimize the steric hindrance of theamino acid binder to COM crystal surface; and synthesizing peptides byvarying the number of spacer amino acids between the binder amino acids.16. The method of claim 15, wherein the peptides are synthesized usingsolid-phase peptide synthesis.
 17. The method of claim 14, wherein thestep of screening the peptide library for high efficacy inhibitorpeptides comprises mixing the test peptide with a supersaturatedsolution of calcium oxalate and measuring inhibition of growth of theCOM crystal using in situ calcium ion-selective electrode measurement.18. The method of claim 14, wherein the molecular level characterizationof the identified peptide is by Atomic Force Microscopy, ScanningElectron Microscopy, and Optical Microscopy.
 19. A method of inhibitingabnormal biomineralization in a subject in need thereof comprisingadministering to the subject therapeutic effective amounts of thecomposition of claim
 1. 20. The method of claim 18, wherein the abnormalbiomineralization causes disease selected from the group consisting ofrecurrent stone disease, primary hyperoxaluria, and systemic oxalosis.